Self-assembling recombinant papillomavirus capsid proteins

ABSTRACT

Recombinant papillomavirus capsid proteins that are capable of self-assembly into capsomer structures and viral capsids that comprise conformational antigenic epitopes are provided. The capsomer structures and viral capsids, consisting of the capsid proteins that are expression products of a bovine, monkey or human papillomavirus L1 conformational coding sequence proteins, can be prepared as vaccines to induce a high-titer neutralizing antibody response in vertebrate animals. The self assembling capsid proteins can also be used as elements of diagnostic immunoassay procedures for papillomavirus infection.

This application is a division of U.S. patent application Ser. No.08/032,869, filed Mar. 16, 1993, now U.S. Pat. No. 5,437,951, which is acontinuation-in-part of U.S. patent application Ser. No. 07/941,371,filed Sep. 3, 1992.

That application is hereby incorporated by reference as if fully setforth herein. The invention relates to recombinant viral proteins. Itrelates particularly to recombinant viral proteins that are suitable foruse in the diagnosis, prophylaxis and therapy of viral infections.

Papillomaviruses infect the epithelia of a wide variety of species ofanimals, including humans, generally inducing benign epithelial andfibro-epithelial tumors, or warts, at the site of infection. Eachspecies of vertebrate is infected by a distinct group ofpapillomaviruses, each papillomavirus group comprising severalpapillomavirus types. For example, more than 60 different humanpapillomavirus (HPV) genotypes have been isolated. Papillomaviruses arehighly species specific infective agents; for example, a bovinepapillomavirus cannot induce papillomas in a heterologous species, suchas humans. Papillomavirus types ALSO appear to be highly specific asimmunogens in that a neutralizing immunity to infection against onepapillomavirus type does not usually confer immunity against anothertype, even when the types infect an homologous species.

In humans, genital warts, which are caused by human papillomaviruses,represent a sexually transmitted disease. Genital warts are very common,and subclinical, or inapparent HPV infection is even more common thanclinical infection. Some benign lesions in humans, particularly thosearising from certain papillomavirus types, undergo malignantprogression. For that reason, infection by one of the malignancyassociated papilloma virus types is considered one of the mostsignificant risk factors in the development of cervical cancer, thesecond most common cancer of women worldwide (zur Hausen, H., 1991;Schiffman, M. 1992). Several different HPV genotypes have been found incervical cancer, with HPV16 being the most common type that is isolatedfrom 50% of cervical cancers.

Immunological studies demonstrating the production of neutralizingantibodies to papillomavirus antigens indicate that papillomavirusinfections and malignancies associated with these infections invertebrate animals could be prevented through immunization; however thedevelopment of effective papillomavirus vaccines has been impeded by anumber of difficulties.

First, it has not been possible to generate in vitro the large stocks ofinfectious virus required to determine the structural and immunogenicfeatures of papillomavirus that are fundamental to the development ofeffective vaccines. Cultured cells express papillomavirus oncoproteinsand other non-structural proteins and these have been extensivelystudied in vitro; but expression of the structural viral proteins, L1and L2 (and the subsequent assembly of infectious virus) occurs only interminally differentiated layers of infected epithelial tissues.Therefore, the characterization of viral genes, proteins, and structurehas necessarily been assembled from studies of virus harvested frompapillomas. In particular, papillomavirus structure and related immunityhave been carried out in the bovine papillomavirus system because largeamounts of infectious virus particles can be isolated from bovinepapillomavirus (BPV) warts.

The information derived from studies of papillomavirus structure to dateindicates that all papillomaviruses are non-enveloped 50-60 nmicosahedral structures (Crawford, L., et al., 1963) which are comprisedof conserved L1 major capsid protein and less well conserved L2 minorcapsid protein (Baker, C., 1987). There is no sequence relationshipbetween the two proteins. The function and location of L2 in the capsidis unclear; however immunologic data suggests that most of L2 isinternal to L1.

Recently, high resolution cryoelectron microscopic analysis of BPV1 andHPV1 virions has determined that the two viruses have a very similarstructure, with 72 pentameric capsomers, each capsomer presumablycomposed of five L1 molecules, forming a virion shell with T=7 symmetry(Baker, T., 1991). The location of the minor L2 capsid protein in thevirion has not been determined, and it is not certain whether L2 orother viral proteins are needed for capsid assembly. Superficially,papillomavirus structure resembles that of the polyoma 45 nm virion,which has the same symmetry and capsomere number (Liddington, R., etal., 1991); however, the systems of intracapsomer contact forpolyomavirus and papillomavirus species are different, and the major andminor capsid proteins of polyomavirus are not genetically related to L1and L2.

Bovine papillomavirus studies are facilitated by a quantitative focaltransformation infectivity assay developed for BPV that is not availablefor HPV (Dvoretzky, I., et al., 1980), and an understanding of immunityto papillomavirus has therefore also been derived from the bovinepapillomavirus system. Limited studies using intact bovinepapillomavirus demonstrated that the non-cutaneous inoculation ofinfectious or formalin-inactivated BPV virus was effective as a vaccineto prevent experimental BPV infection in calves (Olson, C., et al.,1960; Jarrett, W., et al., 1990). Unfortunately, BPV virions cannot beused to develop vaccines against papillomavirus which infects otherspecies, or even vaccines against other bovine types, because of thegreat specificity of these viruses, as well as concern for the oncogenicpotential of intact viral particles.

A significant conclusion of studies of papillomavirus immunity is thatthe ability of antibodies to neutralize papilloma virus appears to berelated to their ability to react with type-specific, conformationallydependent epitopes on the virion surface. For example, rabbit antiseraraised against infectious BPV1 virions inhibits focal transformation ofC127 cells (Doretzky, I., et al., 1980), as well as the transformationof fetal bovine skin grafts; whereas antisera raised against denaturedvirions does not (Ghim, S., et al., 1991).

In contrast, neutralizing sera generated against bacterially derived BPVL1 and L2 (Pilacinski, W. et al., 1984; Jin, X., et al., 1989) andagainst in vitro synthesized cottontail rabbit papillomavirus (CRPV) L1and L2 (Christensen, N., et al., 1991; Lin, Y-L, et al., 1992), neitherof which has the structural features of native virions, had low titers,and the use of recombinant HPV L1 fusion peptides expressed in E. colito detect cellular immune reactivity has had only limited success(Hopfl, R. et al., 1991). The results in the BPV system are consistentwith those of the HPV system, in which monoclonal antibodies thatneutralized HPV11 infection in a mouse xenograft assay recognizednative, but not denatured, HPV11 virions (Christensen, N., et al.,1990).

There have been isolated attempts to produce papillomavirus capsids invitro. Zhou, J. et al. (1991) and (1992) produced virus-like particlesby cloning HPV L1 and L2 genes, and HPV L1 and L2 genes in combinationwith HPV E3/E4 genes into a vaccinia virus vector and infecting CV-1mammalian cells with the recombinant vaccinia virus. These studies wereinterpreted by Zhou to establish that expression of HPV16 L1 and L2proteins in epithelial cells is necessary and sufficient to allowassembly of virion type particles. Cells infected with doublyrecombinant vaccinia virus which expressed L1 and L2 proteins showedsmall (40 nm) virus-like particles in the nucleus that appeared to beincompletely assembled arrays of HPV capsomers. Expressing L1 proteinalone, or L2 protein alone, was expressed did not produce virus-likeparticles; cells doubly infected with singly recombinant vaccinia viruscontaining L1 and L2 genes also did not produce particles. Noneutralizing activity was reported.

Ghim et al., (1992) reported that when L1 from HPV1, a non-genital virustype associated mainly with warts on the hands and feet, was expressedin mammalian cells, the L1 protein contained conformational epitopesfound on intact virions. Ghim did not determine if particles wereproduced, nor was it evaluated if the L1 protein might induceneutralizing antibodies. Even more recently, Hagansee, et al. (1993)reported that when L1 from HPV1 was expressed in human cells, itself-assembled into virus-like particles. No neutralizing antibodystudies were performed.

Studies in other virus systems, for example, parvovirus, indicate thatcapsid assembly alone may not confer immunogenicity. Parvovirus VP2, byitself, was able to self-assemble when expressed in insect cells, butonly particles containing both VP1 and VP2 were able to induceneutralizing antibodies (Kajigaya, S., et al., 1991).

It would be advantageous to develop methods for producing renewablepapillomavirus reagents of any selected species and type in cellculture. It would also be beneficial to produce such papillomavirusreagents having the immunity conferring properties of the conformednative virus particles that could be used as a subunit vaccine.

It is therefore the object of the invention to provide these recombinantconformed papillomavirus proteins, as well as methods for theirproduction and use.

SUMMARY OF THE INVENTION

The invention is directed to the diagnosis and prevention ofpapillomavirus infections and their benign and malignant sequelae byproviding recombinant papillomavirus capsid proteins that self assembleto form capsomer structures comprising conformational epitopes that arehighly specific and highly immunogenic. Therefore, according to theinvention there is provided a genetic construct, comprising apapillomavirus L1 conformational coding sequence, inserted into abaculovirus transfer vector, and operatively expressed by a promoter ofthat vector. The papillomavirus L1 conformational coding sequence can beisolated from a bovine, monkey, or human gene. In a preferredembodiment, the papillomavirus L1 conformational coding sequence isisolated from a wild type HPV16 gene. In a particularly preferredembodiment, the papillomavirus L1 conformational coding sequence isSequence ID No. 2. The genetic construct can further comprise apapillomavirus L2 coding sequence.

According to another aspect of the invention there is provided anon-mammalian eukaryotic host cell transformed by the genetic constructsof the invention.

According to yet another aspect of the invention there is provided amethod for producing a recombinant papillomavirus capsid protein,assembled into a capsomer structure or a portion thereof, comprising thesteps of (1) cloning a papillomavirus gene that codes for an L1conformational capsid protein into a transfer vector wherein the openreading frame of said gene is under the control of the promoter of saidvector; (2) transferring the recombinant vector into a host cell,wherein the cloned papillomavirus gene expresses the papillomaviruscapsid protein; and (3) isolating capsomer structures, comprising thepapillomavirus capsid protein, from the host cell. In a preferredembodiment, the cloned papillomavirus gene consists essentially of theconformational L1 coding sequence, and the expressed protein assemblesinto capsomer structures consisting essentially of L1 capsid protein. Inanother preferred embodiment, the cloning step of the method furthercomprises the cloning of a papillomavirus gene coding for L2 capsidprotein, whereby said L1 and L2 proteins are coexpressed in the hostcell, and wherein the isolated capsomer structures comprise L1 and L2capsid proteins; provided that said transfer vector is not a vacciniavirus when said host cell is a mammalian cell. The conformational L1coding sequence can be cloned from a bovine, monkey, or humanpapillomavirus. According to a preferred embodiment, the conformationalL1 coding sequence is cloned from a wild type HPV16 papillomavirus. In aparticularly preferred embodiment, the conformational L1 coding sequenceis Sequence ID No. 2. Also in a preferred embodiment, the host cell intowhich the genetic construct is transfected is an insect cell. Alsopreferred are embodiments wherein the transfer vector is a baculovirusbased transfer vector, and the papillomavirus gene is under the controlof a promoter that is active in insect cells. Accordingly in thisembodiment, the recombinant baculovirus DNA is transfected into Sf-9insect cells, preferably co-transfected with wild-type baculovirus DNAinto Sf-9 insect cells.

In an alternative embodiment of the method of the invention, thetransfer vector is a yeast transfer vector, and the recombinant vectoris transfected into yeast cells.

According to yet another aspect of the invention there is provided avirus capsomer structure, or a portion thereof, consisting essentiallyof papillomavirus L1 capsid protein, produced by the method theinvention. Alternatively, the virus capsomer structure can consistessentially of papillomavirus L1 and L2 capsid proteins, produced by themethod of the invention. In a particularly preferred embodiment, thevirus capsomer structure comprises papillomavirus L1 capsid protein thatis the expression product of an HPV16 L1 DNA cloned from a wild typevirus. The virus capsids or capsomer structures of the invention, orportions or fragments thereof, can consist essentially of papillomavirusL1 capsid protein. Alternatively, these capsids or capsomer structuresor their fragments can consist essentially of wild type HPV16papillomavirus L1 capsid protein.

The virus capsid structures according to any of the methods of theinvention comprise capsid proteins having immunogenic conformationalepitopes capable of inducing neutralizing antibodies against nativepapillomavirus. The capsid proteins can be bovine, monkey or humanpapillomavirus L1 proteins. In a preferred embodiment, thepapillomavirus L1 capsid protein is the expression product of a wildtype HPV16 L1 gene. In a particularly preferred embodiment, the HPV16 L1gene comprises the sequence of Sequence ID No. 2.

According to yet another aspect of the invention there is provided aunit dose of a vaccine, comprising a peptide having conformationalepitopes of a papillomavirus L1 capsid protein, or L1 protein and L2capsid proteins, in an effective immunogenic concentration sufficient toinduce a papillomavirus neutralizing antibody titer of at least about10³ when administered according to an immunizing dosage schedule. In apreferred embodiment, the vaccine comprises an L1 capsid protein whichis an HPV16 capsid protein. In a particularly preferred embodiment, thevaccine comprises an L1 capsid protein that is a wild type HPV16 L1protein.

Use of the L1 open reading frame (ORF) from a wild type HPV16papillomavirus genome, according to the methods of the invention,particularly facilitates the production of preparative amounts ofvirus-like particles on a scale suitable for vaccine use.

According to yet another aspect of the invention, there is provided amethod of preventing or treating papillomavirus infection in avertebrate, comprising the administration of a papillomavirus capsomerstructure or a fragment thereof according to the invention to avertebrate, according to an immunity-producing regimen. In a preferredembodiment, the papillomavirus capsomer structure comprises wild typeHPV16 L1 capsid protein.

The invention further provides a method of preventing or treatingpapillomavirus infection in a vertebrate, comprising the administrationof the papillomavirus capsomer structure of the invention, or a vaccineproduct comprising the capsomer structure to a vertebrate, according toan immunity-producing regimen. In a preferred embodiment, thepapillomavirus vaccine comprises wild type HPV16 L1 capsid protein.

Also within the scope of the invention is a method for immunizing avertebrate against papillomavirus infection, comprising administering tothe vertebrate a recombinant genetic construct of the inventioncomprising a conformational papillomavirus L1 coding sequence, andallowing said coding sequence to be expressed in the cells or tissues ofsaid vertebrate, whereby an effective, neutralizing, immune response topapillomavirus is induced. In a preferred embodiment, the conformationalpapillomavirus L1 coding sequence is derived from human papillomavirusHPV16. In a particularly preferred embodiment, the human papillomavirusHPV16 is a wild type papillomavirus.

According to yet another aspect of the invention, there is provided amethod of detecting humoral immunity to papillomavirus infection in avertebrate comprising the steps of: (a) providing an effectiveantibody-detecting amount of a papillomavirus capsid peptide having atleast one conformational epitope of a papillomavirus capsomer structure;(b) contacting the peptide of step (a) with a sample of bodily fluidfrom a vertebrate to be examined for papillomavirus infection, andallowing papillomavirus antibodies contained in said sample to bindthereto, forming antigen-antibody complexes; (c) separating saidcomplexes from unbound substances; (d) contacting the complexes of step(c) with a detectably labelled immunoglobulin-binding agent; and (e)detecting anti-papillomavirus antibodies in said sample by means of thelabelled immunoglobulin-binding agent that binds to said complexes. In apreferred embodiment of this aspect of the invention, the peptideconsists essentially of papillomavirus L1 capsid protein. According toan alternative embodiment, the peptide consists essentially of theexpression product of a human papillomavirus HPV16. In a particularlypreferred embodiment, the peptide consists essentially of the expressionproduct of a wild type human papillomavirus HPV16 gene, for example, thepeptide can consist essentially of the expression product of Sequence IDNo. 2.

According to yet another aspect of the invention, there is provided amethod of detecting papillomavirus in a specimen from an animalsuspected of being infected with said virus, comprising contacting thespecimen with antibodies having a specificity to one or moreconformational epitopes of the capsid of said papillomavirus, whereinthe antibodies have a detectable signal producing label, or are attachedto a detectably labelled reagent; allowing the antibodies to bind to thepapillomavirus; and determining the presence of papillomavirus presentin the specimen by means of the detectable label.

According to yet another aspect of the invention, there is provided amethod of determining a cellular immune response to papillomavirus in ananimal suspected of being infected with the virus, comprising contactingimmunocompetent cells of said animal with a recombinant wild typepapillomavirus L1 capsid protein, or combined recombinant L1 and L2capsid proteins according to the invention; and assessing cellularimmunity to papillomavirus by means of the proliferative response ofsaid cells to the capsid protein. In a preferred embodiment of thisaspect of the invention, the recombinant papillomavirus protein isintroduced into the skin of the animal.

According to yet another aspect of the invention there is provided apapillomavirus infection diagnostic kit, comprising capsomer structuresconsisting essentially of papillomavirus L1 capsid protein, or capsomerstructures comprising papillomavirus L1 protein and L2 capsid proteins,or antibodies to either of these capsomer structures, singly or incombination, together with materials for carrying out an assay forhumoral or cellular immunity against papillomavirus, in a unit packagecontainer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression of BPV L1 and HPV16 L1 by means ofrecombinant virus as demonstrated by SDS-PAGE analysis of lysates frominfected insect cells.

FIG. 2 shows the conformation of purified recombinant BPV L1 and HPV16L1 capsid proteins as demonstrated by electron microscopy, compared withauthentic BPV-virions.

FIG. 3 shows the titers of neutralizing antisera induced in animalsinoculated with recombinant BPV L1 as compared to antisera againstintact and denatured BPV virions.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that the gene coding for the L1 major capsid proteinof BPV or HPV16, following introduction into host cells by means of anappropriate transfer vector, can express L1 at high levels, and that therecombinant L1 has the intrinsic capacity to self-assemble into emptycapsomer structures that closely resemble those of an intact virion.

Further, the self-assembled recombinant L1 capsid protein of theinvention, in contrast to L1 protein extracted from recombinantbacteria, or denatured virions, has the efficacy of intactpapillomavirus particles in the ability to induce high levels ofneutralizing antiserum that can protect against papillomavirusinfection. The high level of immunogenicity of the capsid proteins ofthe invention implies strong antibody binding properties that make themsensitive agents in serological screening tests to detect and measureantibodies to conformational virion epitopes. Their immunogenicity alsoindicates that the capsid proteins of the invention can also be used ashighly effective vaccines or immunogens to elicit neutralizingantibodies to protect a host animal against infection by papillomavirus.These observations were recently published in Kirnbauer, et al., (1992),and formed the basis of U.S. application Ser. No. 07/941,371.

We have now discovered that the capsid protein L1 expressed by wild typeHPV16 genomes isolated from benign papillomavirus lesions, whenexpressed in the baculovirus system described, will self-assemble withan efficiency heretofore unknown and comparable to that of bovinepapillovirus L1 capsid protein.

The HPV16 L1 Gene Sequences

The source of HPV16 L1 DNA, as disclosed in published studies, forexample, by Zhou, et al. (1991) was the prototype clone, GenBankAccession No. K02718, that had been isolated from a cervical carcinoma(Seedorf, et al., 1985). We have found that L1 from wild type HPV16genome, which differs from the prototype genome by a single pointmutation, will self-assemble into virus-like particles with anefficiency similar to that seen with BPV L1 or BPV L1/L2. Compared withthe self-assembly seen when L1 from the prototype HPV genome is usedwith L2, L1 from a wild-type genome self-assembles at least 100 timesmore efficiently.

To provide genetic insight into the self-assembly efficiency ofdifferent HPV16 L1 expression products, the open reading frames fromHPV16 L1 genes isolated from both benign lesions and lesions associatedwith dysplasia or carcinoma were sequenced.

The analysis detected two errors in the published sequence of thepublished L1 sequence of the prototype strain, as follows:

(1) there should be an insertion of three nucleotides (ATC) between nt6902 and 6903, which results in the insertion of a serine in the L1protein; and

(2) there should be a deletion in the published prototype sequence ofthree nucleotides (GAT), consisting of nt 6952-6954, which deletes anaspartate from the L1 protein sequence. The corrected nucleotidesequence of the prototype HPV16 L1 genome, consisting of nt 5637-7155,is that of Sequence ID No. 1, listed herein.

The numbering of the nucleotide bases in Sequence ID Nos. 1 and 2 isindexed to 1, and the numbering of nucleotide bases of the published HPVsequence, that is from nt 5638-7156, corresponds to those of thesequence listing from 1-1518. The sites referred to in the originalsequence can be thus readily identified by one skilled in the art.

Three other HPV16 L1 genomes, clone 16PAT; and clones 114/16/2 and114/16/11, were sequenced and those sequences compared to that of thecorrected prototype.

Clone 16PAT, kindly provided by Dennis McCance at the University ofRochester School of Medicine, and cloned from a dysplastic(pre-malignant) lesion of the cervix, expresses an L1 that does notself-assemble efficiently.

Clones 114/16/2 and 114/16/11, kindly provided by Matthias Durst of theGerman Cancer Research Center in Heidelburg, were both cloned fromnon-malignant lesions, and both expressed L1 protein that self-assembledefficiently.

Comparison of Genetic Characteristics of HPV16 L1 Associated withDysplasia, Malignant Progression and Benign Lesions

Clone 16PAT, isolated from papillomavirus infected dysplastic lesionsand the prototype HPV16, isolated from malignant cervical carcinoma,both encode Histidine at nt 6242-6244, while clones 2 and 11, isolatedfrom benign papillomavirus infected lesions (like isolates of many otherpapillomavirus) encode Aspartate at this site.

It appears that this single amino acid difference between the prototype,malignancy-associated HPV16 species, and the HPV16 species from benignlesions accounts for the difference in self-assembly efficiency. It islikely that among closely related HPV types, Aspartate at this locus maybe necessary for efficient self-assembly, and that the substitution ofHistidine for Aspartate impairs this ability in the capsid protein. Theimpairment in capsid assembly in malignancy-associated viruses,associated with loss of the conformational epitopes required for theproduction of neutralizing antibodies, may also be linked to a loweredimmunogenicity which would allow the papillomavirus to escape immunecontrol.

Accordingly, HPV16 L1 genes that express capsid protein thatself-assembles efficiently can be obtained by

(1) isolation of the wild type HPV16 L1 open reading frame from benignlesions of papillomavirus infection; or

(2) carrying out a site specific mutation in the prototype sequence atnt 6242-6244 to encode Aspartate.

Recombinant Capsid Protein

The method of the invention provides a means of preparing recombinantcapsid particles for any papillomavirus. Particles consisting of eitherL1 or L2 capsid protein alone, or consisting of both L1 and L2 capsidproteins together can be prepared. L1/L2 capsid protein particles aremore closely related to the composition of native papillomavirusvirions, but L2 does not appear to be as significant as L1 in conferringimmunity, probably because most of L2 is internal to L1 in the capsidstructure. Although L1 can self-assemble by itself, in the absence ofL2, self-assembled L1/L2 capsid protein particles are more closelyrelated to the composition of native papillomavirus virions.Accordingly, particles comprising L1 alone are simpler, while thosecomprising L1/L2 may have an even more authentic structure. Bothself-assembled L1 and L1/L2 particles induce high-titer neutralizingantibodies and may therefore be suitable for vaccine production.Particles comprising L1 capsid protein expressed by a wild type HPVgenome, either as L1 alone or L1 /L2 together, are particularlypreferred.

Production of the recombinant L1, or combined L1/L2, capsid particles iscarried out by cloning the L1 (or L1 and L2) gene(s) into a suitablevector and expressing the corresponding conformational coding sequencesfor these proteins in a eukaryotic cell transformed by the vector. It isbelieved that the ability to form a capsid-like structure is intimatelyrelated to the ability of the capsid protein to generate high-titerneutralizing antibody, and that in order to produce a capsid proteinthat is capable of self-assembling into capsid structures havingconformational epitopes, substantially all of the capsid protein codingsequence must be expressed. Accordingly, substantially all of the capsidprotein coding sequence is cloned. The gene is preferably expressed in aeukaryotic cell system. Insect cells are preferred host cells; however,yeast cells are also suitable as host cells if appropriate yeastexpression vectors are used. Mammalian cells similarly transfected usingappropriate mammalian expression vectors can also be used to produceassembled capsid protein, however, cultured mammalian cells are lessadvantageous because they are more likely than non-mammalian cells toharbor occult viruses which might be infectious for mammals.

According to a preferred protocol, a baculovirus system is used. Thegene to be cloned, substantially all of the coding sequence for bovinepapillomavirus (BPV1) or human papillomavirus (HPV16) L1 capsid protein,or human papillomavirus HPV16 L1 and L2, is inserted into a baculovirustransfer vector containing flanking baculovirus sequences to form a geneconstruct, and the recombinant DNA is co-transfected with wild typebaculovirus DNA into Sf-9 insect cells as described in Example 1, togenerate recombinant virus which, on infection, can express the insertedgene at high levels. The actual production of protein is made byinfecting fresh insect cells with the recombinant baculovirus;accordingly, the L1 capsid protein and the L1 and L2 capsid proteins areexpressed in insect cells that have been infected with recombinantbaculovirus as described in Example 2.

In the procedure of Example 1, the complete L1 gene of BPV1 wasamplified by polymerase chain reaction (PCR; Saiki, R., et al., 1987)and cloned into AcMNPV (Autographa californica nuclear polyhedrosisvirus) based baculovirus vector (Summers, M. et al., 1987). The L1 openreading frame was put under the control of the baculovirus polyhedrinpromoter. After co-transfection of the L1 clone with the wild type (wt)baculovirus DNA into Sf-9 insect cells (ATCC Accession No. CRL 1711) andplaque purification of recombinant clones, high titer recombinant viruswas generated. Extracts-from cells infected with wt AcMNPV or BPV1 L1recombinant viruses (AcBPV-L1) (Example 2) were analyzed bypolyacrylamide gel electrophoresis. After Coomassie blue staining, aunique protein of the predicted size, 55 kilodaltons, was detected inextracts from the cultures infected with the AcBPV1-L1 virus (FIG. 1A).The identity of this protein as BPV L1 was verified by immunoblotting(FIG. 1B), using a BPV L1 specific monoclonal antibody (Nakai, Y., etal., 1986).

To test the hypothesis that papillomavirus L1 has the ability toself-assemble into virus-like particles when overexpressed inheterologous cells, electron micrographs of thin sections from AcBPV-L1infected cells were examined for the presence of papillomavirus-likestructures. Cells infected with the BPV recombinant virus contained manycircular structures of approximately 50 nm which were preferentiallylocalized in the nucleus; these structures were absent from wild typebaculovirus infected cells. These results suggested that self assemblyof L1 into virus-like particles had occurred, since in vivopapillomavirus virion assembly takes place in the nucleus and thediameter of the virions has been reported as 55 nm.

Following expression of the conformed capsid protein in the host cell,virus particles are purified from lysates of infected cells as describedin Example 4. To obtain further evidence that the L1 protein hadself-assembled, virus-like particles were isolated from the infectedinsect cells by means of gradient centrifugation (FIG. 2).

High molecular mass structures were separated from lysates of L1recombinant or wild type infected cells by centrifugation through a 40%sucrose cushion and the pelleted material was subjected to CsCl densitygradient centrifugation. Fractions were collected and tested forreactivity to the BPV L1 specific monoclonal antibody by immunoblotting.

L1 positive fractions from the gradient were adsorbed onto carbon filmgrids, stained with 1% uranyl acetate and examined by transmissionelectron microscopy. The positive fractions contained numerous circularstructures that exhibited a regular array of capsomers (FIG. 2A).Consistent with previous reports of the density of empty BPV virions(Larsen, P., et al., 1987), the density of the CsCl fraction containingthe peak of the virus-like particles was approximately 1.30 gm/ml. Mostwere approximately 50 nm in diameter, although smaller circles andpartially assembled structures were also seen. The larger particles werevery similar in size and subunit structure to infectious BPV virionsthat had been stained and photographed concurrently (FIG. 2B). Theseparticles were not observed in preparations from mock infected or wtAcMNPV infected cells. These results indicate that BPV L1 has theintrinsic capacity to assemble into virus-like particles in the absenceof L2 or other papillomavirus proteins. In addition, specific factorslimited to differentiating epithelia or mammalian cells are not requiredfor papillomavirus capsid assembly.

To determine if the ability to self-assemble in insect cells is ageneral feature of papillomavirus L1, we also expressed the L1 of HPV16,the HPV type most often detected in human genital cancers, via ananalogous recombinant baculovirus. A protein of the expected 58 kd sizewas expressed at high levels in the insect cells infected with theHPV16-L1 recombinant virus (FIG. 1A) and it reacted strongly with anHPV16 L1 monoclonal antibody (which also reacted weakly with BPV L1;FIG. 1C). After CsCl gradient purification, immunoreactive fractionswere examined by electron microscopy and found to contain 50 nmpapillomavirus-like particles (FIG. 2C). Although somewhat fewercompletely assembled particles were seen in the human system incomparison to the BPV L1 preparations, possibly due to the lower levelsof expression or greater extent of HPV16 L1 degradation (FIG. 1), theresults conclusively indicate that the L1 of the HPV16 and presumablythe L1 proteins of other types, have the intrinsic capacity to assembleinto virion-type structures. Preparations of recombinant papillomaviruscapsid particles for Rhesus monkey PV have also been carried out asdescribed in the Examples.

Recombinant Conformed Capsid Proteins as Immunogens

Subunit vaccines, based on self-assembled major capsid proteinssynthesized in heterologous cells, have been proved effective inpreventing infections by several pathogenic viruses, including humanhepatitis B (Stevens, C., et al., 1987).

Studies demonstrating that infectious or formalin inactivated BPV iseffective as a vaccine, while BPV transformed cells are ineffective,suggest that viral capsid proteins, rather than early gene products,elicit the immune response. Other data in the scientific literatureindicates that L1 protein extracted from bacteria was partiallysuccessful in eliciting an immune response despite the low titers ofneutralizing antibodies. Accordingly, the BPV L1 that was expressed andassembled into virus-like particles in insect cells was studied for itsability to induce neutralizing antisera in rabbits. Two types ofpreparations were tested: whole cell extracts of L1 recombinant or wildtype infected Sf-9 cells and partially purified particles isolated bydifferential centrifugation and ammonium sulfate precipitation.Following a primary inoculation, the rabbits received two biweeklybooster inoculations.

The rabbit sera were tested for the ability to inhibit BPV infection ofmouse C127 cells, as measured by a reduction in the number of fociinduced by a standard amount of BPV virus (a representative assay isshown in FIG. 3). The immune sera generated by inoculation withbaculovirus derived L1 were able to reduce the infectivity of the BPVvirus by 50% at a dilution of at least 1:11,000 (a titer of 11,000;Table 1), whereas the preimmune sera from the same rabbits did notinhibit focal transformation at a dilution of 1:20, the lowest dilutiontested. Both the crude preparations and partially purified particleswere effective in inducing high titer neutralizing antisera, with290,000 being the highest titer measured. This was the same as theneutralizing titer of the positive control antiserum raised againstinfectious BPV virions. In comparison, the highest titer generated in aprevious study using bacterially derived L1 was 36 (Pilancinski, W., etal., 1984). The serum from the rabbit inoculated with the extract fromthe wild type baculovirus infected cells was unable to inhibitinfectivity at a dilution of 1:20, indicating that the neutralizingactivity was L1 specific. Disruption of the partially purified L1particles, by boiling in 1% SDS, abolished the ability of thepreparation to induce neutralizing antibodies (Table 1). Thedemonstration that L1 can self-assemble into virion-like particles thatelicit neutralizing antisera titers at least three orders of magnitudehigher than previous in vitro-produced antigens suggests the recombinantL1 capsid proteins has the potential to induce effective long termprotection against naturally transmitted papillomavirus. In view ofthese results, it appears that the L1 particles assembled in insectcells mimic infectious virus in the presentation of conformationallydependent immunodominant epitopes. These results also establish that L2is not required for the generation of high titer neutralizingantibodies. The reported weak neutralizing immunogenicity of bacteriallyderived L1 may occur because it does not assume an appropriateconformation or has not assembled into virion like structures. Also,multiple electrophoretic variants of L1 have been detected in virions(Larsen, P., et al., 1987). Some of these modified species, which areprobably absent in the bacterially derived L1, may facilitate thegeneration of neutralizing antibodies.

The ability of recombinant L1 (or L2) papillomavirus capsid proteinssuch as those disclosed herein to induce high titer neutralizingantiserum makes them suitable for use as vaccines for prophylaxisagainst communicable papillomatosis. Examples of populations at riskthat could benefit from immunization are bovine herds, which aresusceptible to papilloma warts; all humans for non-genital types of HPVinfection; and sexually active humans for genital HPV types ofinfection.

Therapeutic vaccination can be useful for productive papillomaviruslesions, which usually express L1 (and L2) capsid proteins. Such lesionsare most likely to occur in benign infections, such as warts orlaryngeal papillomatosis. Laryngeal papillomatosis in newborns isusually contracted by the infant during passage through the birth canalwhere infectious papillomavirus is present in vaginal secretions.Therapeutic vaccination of infected pregnant women against thepapillomavirus can induce neutralizing IgG antibody capable of passingthrough the placental barrier and into the circulation of the fetus toprovide prophylactic passive immunity in the infant against this type ofpapillomavirus infection. Additional infant-protecting mechanisms areprovided by maternal IgA which is secreted into the vaginal fluid andinto breast milk. Jarrett (1991) demonstrates some therapeutic efficacyfor L2 in treating BPV-induced warts. Malignant tumors typically do notexpress L1 or L2, and the efficacy of vaccination with recombinant L1 orL2 in conditions such as cervical cancer, is uncertain.

Protective immunity against both benign and malignant papillomavirusdisease can be induced by administering an effective amount ofrecombinant L1 capsid protein to an individual at risk forpapillomavirus infection. A vaccine comprising the capsid protein can bedirectly administered, either parenterally or locally, according toconventional immunization protocols. In an alternative embodiment, theconformational coding sequence of L1 can be cloned into a transfervector, for example, a semliki forest virus vector (which produces amild transient infection), the recombinant virus introduced into thecells or tissues of the recipient where the immunizing capsid protein isthen expressed. Vaccinia virus can also be used as a vehicle for thegene.

Recombinant Conformed Capsid Proteins as Serological Screening Agents

Published serologic studies of human immune response to papillomavirusvirion proteins have principally utilized bacterially derived L1 and L2capsid proteins, and the results have not correlated well with othermeasures of HPV infection (Jenison, S., et al., 1990). BPVpapillomavirus immunity studies described above indicate thatpapillomavirus virion proteins extracted from bacteria do not presentthe conformationally dependent epitopes that appear to be type-specificand recognized by most neutralizing antibodies. Compared with suchassays that primarily recognize linear epitopes, a serological testusing self-assembled L1 particles is likely to be a more accuratemeasure of the extent of anti-HPV virion immunity in the humanpopulation. The recombinant L1 capsid proteins disclosed herein,presenting conformational epitopes, can therefore be used as highlyspecific diagnostic reagents to detect immunity conferring neutralizingantibody to papilloma virus in binding assays of several types. Theprocedures can be carried out generally as either solid phase orsolution assays that provide a means to detect antibodies in bodilyfluids that specifically bind to the capsid protein in antigen-antibodypairs. Examples of procedures known to those skilled in the art forevaluating circulating antibodies are solution phase assays, such asdouble-antibody radioimmunoassays or enzyme immunoassays, or solid phaseassays such as strip radioimmunoassay based on Western blotting or anenzyme-linked immunoabsorbent assay (ELISA) as disclosed in U.S. Pat.No. 4,520,113 to Gallo et al., or immunochromatographic assays asdisclosed in U.S. Pat. No. 5,039,607 to Skold et al. A preferred ELISAmethod for the detection of antibodies is that disclosed in Harlow, E.,and Lane, D. in Antibodies: A Laboratory Manual Cold Spring Harbor,N.Y., 1988, pp. 563-578.

The recombinant L1 or L1/L2 capsid proteins disclosed herein can also beused to measure cellular immunity to papillomavirus by means of in vivoor in vitro assays, for example, antigen-induced T-cell proliferativeresponses as described by Bradley, L., 1980, and particularly cellularresponses to viral antigens, as described in U.S. Pat. No. 5,081,029 toStarling. Cellular immunity to papillomavirus can also be determined bythe classical in vivo delayed hypersensitivity skin test as described byStites, D., 1980; or in a preferred method, according to Hopfl, R., etal., 1991, by the intradermal injection of recombinant HPV L1 fusionproteins.

The capsid proteins of the invention can also be used as immunogens toraise polyclonal or monoclonal antibodies, according to methods wellknown in the art. These papillomavirus-specific antibodies, particularlyin combination with labelled second antibodies, specific for a class orspecies of antibodies, can be used diagnostically according to variousconventional assay procedures, such as immunohistochemistry, to detectthe presence of capsid proteins in samples of body tissue or bodilyfluids.

The genetic manipulations described below are disclosed in terms oftheir general application to the preparation of elements of the geneticregulatory unit of the invention. Occasionally, the procedure may not beapplicable as described to each recombinant molecule included within thedisclosed scope. The situations for which this occurs will be readilyrecognized by those skilled in the art. In all such cases, either theoperations can be successfully performed by conventional modificationsknown to those skilled in the art, e.g. by choice of an appropriatealternative restriction enzyme, by changing to alternative conventionalreagents, or by routine modification of reaction conditions.Alternatively, other procedures disclosed herein or otherwiseconventional will be applicable to the preparation of the correspondingrecombinant molecules of the invention. In all preparative methods, allstarting materials are known or readily preparable from known startingmaterials. In the following examples, all temperatures are set forth indegrees Celsius; unless otherwise indicated, all parts and percentagesare by weight.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the invention to itsfullest extent. The following preferred embodiments are therefore to beconstrued as merely illustrative and not limiting the remainder of thedisclosure in any way whatsoever.

EXAMPLE 1

Full length L1, or L1 and L2 open reading frames (ORF) were amplified byPCR using the cloned prototypes of BPV1 DNA (Chen, E., et al., 1982),GenBank Accession No. X02346 or HPV16 DNA (Seedorf, K., et al., 1985),GenBank Accession No. K02718; or wild type HPV16 DNA (Sequence ID No. 2)as templates. Unique restriction sites were incorporated into theoligonucleotide primers (underlined).

BPV1-L1 primer sequence (Sequence ID No. 3):

5'-CCGCTGAATTCAATATGGCGTTGTGGCAACAAGGCCAGAAGCTGTAT-3' (sense) and(Sequence ID No. 4):

5'-GCGGTGGTACCGTGCAGTTGACTTACCTTCTGTTTTACATTTACAGA-3' (antisense);

HPV16-L1 primer sequence (Sequence ID No. 5):

5'-CCGCTAGATCTAATATGTCTCTTTGGCTGCCTAGTGAGGC('-3' (sense); and (SequenceID No. 6):

5'-GCGGTAGATCTACACTAATTCAACATACATACAATACTTACAGC-3' (antisense).

L1 coding sequences begin at the 1st methionine codon (bold) for BPV1and the 2nd methionine for HPV16. BPV1-L1 was cloned as a 5'-EcoRI to3'-KpnI fragment and HPV16-L1 as a 5'-BglII to 3'-BglII fragment intothe multiple cloning site downstream of the polyhedrin promoter of theACMNPV based baculovirus transfer vector pEV mod (Wang, X., et al. 1991)and verified by sequencing through the AcMNPV/L1 junction. A quantity of2 μg of CsCl-purified recombinant plasmid was cotransfected with 1 μgwild type AcMNPV DNA (Invitrogen, San Diego, Calif.) into Sf-9 cells(ATCC) using lipofectin (Gibco/BRL, Gaithersburg, Md.) (Hartig, P., etal., 1991) and the recombinant baculoviruses plaque-purified asdescribed (Summers, M., et al., 1987).

EXAMPLE 2 Expression of L1 Proteins or L1/L2 proteins in Insect Cells

Sf-9 cells were either mock infected (mock) or infected at amultiplicity of infection of 10 with either wt AcMNPV (wt) or AcBPV-L1(B-L1), AcHPV16-L1 (16-L1), or AcHPV16-L1 (16-L1) and AcHPV16-L2 (16-L2)recombinant virus. After 72 hours, cells were lysed by boiling inLaemmli buffer and the lysates subjected to SDS-PAGE in 10% gels.Proteins were either stained with 0.25% Coomassie blue (FIG. 1A) orimmunoblotted and probed with BPV L1 mAb AU-1 (Nakai, Y., et al.,1986)(Fig. 1B) or HPV16L1 mAb CAMVIR-1 (McLean, C., et al., 1990) (FIG.1C) and ¹²⁵ I-labeled Fab anti-mouse IgG (Amersham). P designatespolyhedrin protein.

EXAMPLE 3 Production of Antisera

Rabbits were immunized by subcutaneous injection either with whole cellSf-9 lysates (3×10⁷ cells) prepared by one freeze/thaw cycle and 20×dounce homogenization (rabbit #1,2, and 8) or with 200 μg of L1 proteinpartially purified by differential centrifugation and 35% ammoniumsulfate precipitation (#3,4,6, and 7), in complete Freund's adjuvant,and then boosted twice at two week intervals, using the samepreparations in incomplete Freund's adjuvant.

EXAMPLE 4 Purification of Particles and Transmission ElectronMicroscopic (EMK) Analysis

500 ml of Sf-9 cells (2×10⁶ /ml) were infected with AcBPV-L1 (FIG. 2A)or AcHPV16-L1 (FIG. 2C) or or AcHPV16-L1/L2 (16-L1/L2) recombinantbaculoviruses. After 72 hr, the harvested cells were sonicated in PBSfor 60 sec. After low speed clarification, the lysates were subjected tocentrifugation at 110,000 g for 2.5 hr through a 40% (wt/vol)sucrose/PBS cushion (SW-28). The resuspended pellets were centrifuged toequilibrium at 141,000 g for 20 hr at room temperature in a 10-40%(wt/wt) CsCl/PBS gradient. Fractions were harvested from the bottom andanalyzed by SDS-PAGE. Immunoreactive fractions were dialyzed againstPBS, concentrated by Centricon 30 (Millipore) ultrafiltration, and (forHPV16-L1) pelleted by centrifugation for 10 min at 30 psi in a A-100rotor in an airfuge (Beckman). BPV1 virions (FIG. 2B) were purified froma bovine wart (generously provided by A. B. Jenson) as described(Cowsert, L., et al., 1987). Purified particles were adsorbed to carboncoated TEM grids, stained with 1% uranyl acetate and examined with aPhilips electron microscope EM 400T at 36,000× magnification. Resultsare shown in FIG. 2. The bar=50 nm!.

EXAMPLE 5 BPV1 Neutralization Assay

Serial dilutions of sera obtained 3 wk after the second boost wereincubated with approximately 500 focus forming units of BPV1 virus for30 min, the virus absorbed to C127 cells for 1 hr and the cells culturedfor 3 weeks (Dvoretzky, I., et al., 1980). The foci were stained with0.5% methylene blue/0.25% carbol fuchsin/methanol. The results are shownin FIG. 3 and are discussed below. The antisera and dilutions used areindicated below the plates. Anti-AcBPV-L1 was obtained from rabbit #1and anti-wt ACMNPV from rabbit #8 (Table 1). The normal rabbit serumnegative control is designated "nrs"; anti-BPV-1 virion was raisedagainst native BPV virions in a previous study (Nakai, Y., et al.,1986); and Dako is the commercially available (Dako Corp., SantaBarbara, Calif.) rabbit antiserum raised against denatured BPV virions.

EXAMPLE 6 Serum Neutralizing Titer against BPV1

Assays were carried out as in Example 5. Rabbits #1, 2, and 8 wereinoculated with crude whole cell Sf-9 lysates, and rabbits # 3,4,6, and7 with partially purified L1 protein (Table 1). Rabbits #6 and 7 wereimmunized with L1 protein preparations that had been denatured byboiling in 1% SDS. At least two bleeds, taken 3-6 weeks after the secondboost, were tested for each rabbit and found to have the same titer. Thetiter of the preimmune sera from each of the rabbits was less than 20,the lowest dilution tested.

                  TABLE 1                                                         ______________________________________                                                          serum neutralization titer                                               rabbit                                                                             against BPV1*                                               ______________________________________                                        AcBPV-L1       1      11,000                                                  "              2      97,000                                                  "              3      290,000                                                 "              4      97,000                                                  BPV1-virions†                                                                         5      290,000                                                 AcBPV-L1/SDS   6      <2                                                      "              7      <2                                                      wt AcMNPV      8      <20                                                     ______________________________________                                         reciprocal of dilution that caused 50% focus reduction                        †provided by A. B. Jenson (Nakai, Y., et al., 1986).              

The invention may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive, and the scope of the invention is, therefore, indicated bythe appended claims rather than by the foregoing description. Allmodifications which come within the meaning and range of the lawfulequivalency of the claims are to be embraced within their scope.

BIBLIOGRAPHY

U.S. Pat. No. 5,081,029 to Starling et al.

U.S. Pat. No. 5,039,607 to Skold et al.

U.S. Pat. No. 4,520,113 to Gallo et al.

Baker, C. in The Papovaviridae: Vol.2. The Papillomaviruses (N. Salzmanet al., eds.) Plenum Press, New York, 1987. p.321.

Baker, T., et al. Biophys. J. 60:1445 (1991).

Bradley, L. et al. in Selected Methods in Cellular Immunology. B.Mishell and S. Shiigi, eds. San Francisco: W. H. Freeman and Co., 1980.pp. 164-166.

Christensen, N., et al. Virology 64:5678 (1990).

Christensen, N., et al. Virology 181:572 (1991).

Crawford, L., et al. Virology 21:258 (1963).

Dvoretzky, I., et al. Virology 103:369 (1980).

Ghim, S., et al. Comparison of neutralization of BPV-1 infection of C127cells and bovine fetal skin xenografts. Int. J. Cancer 49: 285 (1991).

Ghim, S., et al. HPV1-L1 protein expressed in cos cells displaysconformational epitopes found on intact virions. Virology 190:548-552(1992).

Hagensee, M., et al. Self-assembly of human papillomavirus type 1capsids by expression of the L1 protein alone or by coexpression of theL1 and L2 capsid proteins. J. of Virology 67(1):315-322.

Hopfl, R., et al. Skin test for HPV type 16 proteins in cervicalintraepithelial neoplasia. Lancet 337:373 (1991).

Jarrett, W., et al. Veterinary Record 126:449 (1990).

Jarrett, W., et al. Studies on vaccination against papillomaviruses:prophylactic and therapeutic vaccination with recombinant structuralproteins. Virology 184:33 (1991).

Jenison, S., et al. J. Infectious Dis. 162:60 (1990).

Jenson, A., et al. Identification of linear epitopes BPV-1 L1 proteinrecognized by sera of infected or immunized animals. Pathobiology 59:396(1991)

Jin, X., et al. J. Gen. Virology 70:1133 (1989). Kajigaya, S., et al.Proc. Natl. Acad. Sci. USA 88:4646 (1991).

Kirnbauer, R., et al. Papillomavirus L1 major capsid proteinself-assembles into virus-like particles that are highly immunogenic.Proc. Natl. Acad. Sci. USA 89:12180-12184 (1992).

Larsen, P., et al. J. Virology 61:3596 (1987).

Liddington, R., et al. Nature 354:278 (1991).

Lin, Y-L., et al. Effective vaccination against papilloma development byimmunization with L1 or L2 structural protein of cottontail rabbitpapillovirus. Virology 187:612 (1992).

McLean, C., et al. Production and characterization of a monoclonalantibody to human papillomavirus type 16 using recombinant vacciniavirus. J. Clin. Pathol 43:488 (1990).

Nakai, Y. Intervirol. 25:30 (1986).

Olson, C., et al. Amer. J. Vet. Res. 21:233 (1960).

Pilacinski, W., et al. Biotechnology 2:356 (1984).

Saiki, R. K., et al. Science 239:487 (1987).

Seedorf, et al. Human papillomavirus type 16 DNA seqeunce. Virology145:181-185 (1985)

Shiffman, M. J. National Cancer Inst. 84:394 (1992).

Stevens, C., et al. JAMA 257:2612 (1987).

Stites, D. Chapter 27 in Basic and Clinical Immunology 3d Ed. H.Fudenberg et al., eds. Los Altos: Lange Medical Publications, 1980.

Summers, M., et al. Texas Agricultural Experiment Station, CollegeStation, Tex. A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures (1987). Bulletin No. 1555.

Zhou, J., et al. Expression of vaccinia recombinant HPV 16 L1 and L2 ORFproteins in epithelial cells is sufficient for assembly of HPVvirion-like particles. J. Virology 185:251 (1991).

zur Hausen, H. Science 254:1167 (1991).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1517 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Human papillomavirus                                            (B) STRAIN: HPV16                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1517                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGTCTCTTTGGCTGCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTC48                            MetSerLeuTrpLeuProSerGluAlaThrValTyrLeuProProVal                              151015                                                                        CCAGTATCTAAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAAC96                            ProValSerLysValValSerThrAspGluTyrValAlaArgThrAsn                              202530                                                                        ATATATTATCATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCC144                           IleTyrTyrHisAlaGlyThrSerArgLeuLeuAlaValGlyHisPro                              354045                                                                        TATTTTCCTATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAA192                           TyrPheProIleLysLysProAsnAsnAsnLysIleLeuValProLys                              505560                                                                        GTATCAGGATTACAATACAGGGTATTTAGAATACATTTACCTGACCCC240                           ValSerGlyLeuGlnTyrArgValPheArgIleHisLeuProAspPro                              65707580                                                                      AATAAGTTTGGTTTTCCTGACACCTCATTTTATAATCCAGATACACAG288                           AsnLysPheGlyPheProAspThrSerPheTyrAsnProAspThrGln                              859095                                                                        CGGCTGGTTTGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCA336                           ArgLeuValTrpAlaCysValGlyValGluValGlyArgGlyGlnPro                              100105110                                                                     TTAGGTGTGGGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGAC384                           LeuGlyValGlyIleSerGlyHisProLeuLeuAsnLysLeuAspAsp                              115120125                                                                     ACAGAAAATGCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGA432                           ThrGluAsnAlaSerAlaTyrAlaAlaAsnAlaGlyValAspAsnArg                              130135140                                                                     GAATGTATATCTATGGATTACAAACAAACACAATTGTGTTTAATTGGT480                           GluCysIleSerMetAspTyrLysGlnThrGlnLeuCysLeuIleGly                              145150155160                                                                  TGCAAACCACCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACC528                           CysLysProProIleGlyGluHisTrpGlyLysGlySerProCysThr                              165170175                                                                     AATGTTGCAGTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAAC576                           AsnValAlaValAsnProGlyAspCysProProLeuGluLeuIleAsn                              180185190                                                                     ACAGTTATTCAGGATGGTGATATGGTTCATACTGGCTTTGGTGCTATG624                           ThrValIleGlnAspGlyAspMetValHisThrGlyPheGlyAlaMet                              195200205                                                                     GACTTTACTACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATT672                           AspPheThrThrLeuGlnAlaAsnLysSerGluValProLeuAspIle                              210215220                                                                     TGTACATCTATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAA720                           CysThrSerIleCysLysTyrProAspTyrIleLysMetValSerGlu                              225230235240                                                                  CCATATGGCGACAGCTTATTTTTTTATTTACGAAGGGAACAAATGTTT768                           ProTyrGlyAspSerLeuPhePheTyrLeuArgArgGluGlnMetPhe                              245250255                                                                     GTTAGACATTTATTTAATAGGGCTGGTACTGTTGGTGAAAATGTACCA816                           ValArgHisLeuPheAsnArgAlaGlyThrValGlyGluAsnValPro                              260265270                                                                     GACGATTTATACATTAAAGGCTCTGGGTCTACTGCAAATTTAGCCAGT864                           AspAspLeuTyrIleLysGlySerGlySerThrAlaAsnLeuAlaSer                              275280285                                                                     TCAAATTATTTTCCTACACCTAGTGGTTCTATGGTTACCTCTGATGCC912                           SerAsnTyrPheProThrProSerGlySerMetValThrSerAspAla                              290295300                                                                     CAAATATTCAATAAACCTTATTGGTTACAACGAGCACAGGGCCACAAT960                           GlnIlePheAsnLysProTyrTrpLeuGlnArgAlaGlnGlyHisAsn                              305310315320                                                                  AATGGCATTTGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACT1008                          AsnGlyIleCysTrpGlyAsnGlnLeuPheValThrValValAspThr                              325330335                                                                     ACACGCAGTACAAATATGTCATTATGTGCTGCCATATCTACTTCAGAA1056                          ThrArgSerThrAsnMetSerLeuCysAlaAlaIleSerThrSerGlu                              340345350                                                                     ACTACATATAAAAATACTAACTTTAAGGAGTACCTACGACATGGGGAG1104                          ThrThrTyrLysAsnThrAsnPheLysGluTyrLeuArgHisGlyGlu                              355360365                                                                     GAATATGATTTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACT1152                          GluTyrAspLeuGlnPheIlePheGlnLeuCysLysIleThrLeuThr                              370375380                                                                     GCAGACGTTATGACATACATACATTCTATGAATTCCACTATTTTGGAG1200                          AlaAspValMetThrTyrIleHisSerMetAsnSerThrIleLeuGlu                              385390395400                                                                  GACTGGAATTTTGGTCTACAACCTCCCCCAGGAGGCACACTAGAAGAT1248                          AspTrpAsnPheGlyLeuGlnProProProGlyGlyThrLeuGluAsp                              405410415                                                                     ACTTATAGGTTTGTAACATCCCAGGCAATTGCTTGTCAAAAACATACA1296                          ThrTyrArgPheValThrSerGlnAlaIleAlaCysGlnLysHisThr                              420425430                                                                     CCTCCAGCACCTAAAGAAGATCCCCTTAAAAAATACACTTTTTGGGAA1344                          ProProAlaProLysGluAspProLeuLysLysTyrThrPheTrpGlu                              435440445                                                                     GTAAATTTAAAGGAAAAGTTTTCTGCAGACCTAGATCAGTTTCCTTTA1392                          ValAsnLeuLysGluLysPheSerAlaAspLeuAspGlnPheProLeu                              450455460                                                                     GGACGCAAATTTTTACTACAAGCAGGATTGAAGGCCAAACCAAAATTT1440                          GlyArgLysPheLeuLeuGlnAlaGlyLeuLysAlaLysProLysPhe                              465470475480                                                                  ACATTAGGAAAACGAAAAGCTACACCCACCACCTCATCTACCTCTACA1488                          ThrLeuGlyLysArgLysAlaThrProThrThrSerSerThrSerThr                              485490495                                                                     ACTGCTAAACGCAAAAAACGTAAGCTGTA1517                                             ThrAlaLysArgLysLysArgLysLeu                                                   500505                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1517 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1517                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ATGTCTCTTTGGCTGCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTC48                            MetSerLeuTrpLeuProSerGluAlaThrValTyrLeuProProVal                              151015                                                                        CCAGTATCTAAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAAC96                            ProValSerLysValValSerThrAspGluTyrValAlaArgThrAsn                              202530                                                                        ATATATTATCATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCC144                           IleTyrTyrHisAlaGlyThrSerArgLeuLeuAlaValGlyHisPro                              354045                                                                        TATTTTCCTATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAA192                           TyrPheProIleLysLysProAsnAsnAsnLysIleLeuValProLys                              505560                                                                        GTATCAGGATTACAATACAGGGTATTTAGAATACATTTACCTGACCCC240                           ValSerGlyLeuGlnTyrArgValPheArgIleHisLeuProAspPro                              65707580                                                                      AATAAGTTTGGTTTTCCTGACACCTCATTTTATAATCCAGATACACAG288                           AsnLysPheGlyPheProAspThrSerPheTyrAsnProAspThrGln                              859095                                                                        CGGCTGGTTTGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCA336                           ArgLeuValTrpAlaCysValGlyValGluValGlyArgGlyGlnPro                              100105110                                                                     TTAGGTGTGGGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGAC384                           LeuGlyValGlyIleSerGlyHisProLeuLeuAsnLysLeuAspAsp                              115120125                                                                     ACAGAAAATGCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGA432                           ThrGluAsnAlaSerAlaTyrAlaAlaAsnAlaGlyValAspAsnArg                              130135140                                                                     GAATGTATATCTATGGATTACAAACAAACACAATTGTGTTTAATTGGT480                           GluCysIleSerMetAspTyrLysGlnThrGlnLeuCysLeuIleGly                              145150155160                                                                  TGCAAACCACCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACC528                           CysLysProProIleGlyGluHisTrpGlyLysGlySerProCysThr                              165170175                                                                     AATGTTGCAGTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAAC576                           AsnValAlaValAsnProGlyAspCysProProLeuGluLeuIleAsn                              180185190                                                                     ACAGTTATTCAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATG624                           ThrValIleGlnAspGlyAspMetValAspThrGlyPheGlyAlaMet                              195200205                                                                     GACTTTACTACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATT672                           AspPheThrThrLeuGlnAlaAsnLysSerGluValProLeuAspIle                              210215220                                                                     TGTACATCTATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAA720                           CysThrSerIleCysLysTyrProAspTyrIleLysMetValSerGlu                              225230235240                                                                  CCATATGGCGACAGCTTATTTTTTTATTTACGAAGGGAACAAATGTTT768                           ProTyrGlyAspSerLeuPhePheTyrLeuArgArgGluGlnMetPhe                              245250255                                                                     GTTAGACATTTATTTAATAGGGCTGGTACTGTTGGTGAAAATGTACCA816                           ValArgHisLeuPheAsnArgAlaGlyThrValGlyGluAsnValPro                              260265270                                                                     GACGATTTATACATTAAAGGCTCTGGGTCTACTGCAAATTTAGCCAGT864                           AspAspLeuTyrIleLysGlySerGlySerThrAlaAsnLeuAlaSer                              275280285                                                                     TCAAATTATTTTCCTACACCTAGTGGTTCTATGGTTACCTCTGATGCC912                           SerAsnTyrPheProThrProSerGlySerMetValThrSerAspAla                              290295300                                                                     CAAATATTCAATAAACCTTATTGGTTACAACGAGCACAGGGCCACAAT960                           GlnIlePheAsnLysProTyrTrpLeuGlnArgAlaGlnGlyHisAsn                              305310315320                                                                  AATGGCATTTGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACT1008                          AsnGlyIleCysTrpGlyAsnGlnLeuPheValThrValValAspThr                              325330335                                                                     ACACGCAGTACAAATATGTCATTATGTGCTGCCATATCTACTTCAGAA1056                          ThrArgSerThrAsnMetSerLeuCysAlaAlaIleSerThrSerGlu                              340345350                                                                     ACTACATATAAAAATACTAACTTTAAGGAGTACCTACGACATGGGGAG1104                          ThrThrTyrLysAsnThrAsnPheLysGluTyrLeuArgHisGlyGlu                              355360365                                                                     GAATATGATTTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACT1152                          GluTyrAspLeuGlnPheIlePheGlnLeuCysLysIleThrLeuThr                              370375380                                                                     GCAGACGTTATGACATACATACATTCTATGAATTCCACTATTTTGGAG1200                          AlaAspValMetThrTyrIleHisSerMetAsnSerThrIleLeuGlu                              385390395400                                                                  GACTGGAATTTTGGTCTACAACCTCCCCCAGGAGGCACACTAGAAGAT1248                          AspTrpAsnPheGlyLeuGlnProProProGlyGlyThrLeuGluAsp                              405410415                                                                     ACTTATAGGTTTGTAACCCAGGCAATTGCTTGTCAAAAACATACACCT1296                          ThrTyrArgPheValThrGlnAlaIleAlaCysGlnLysHisThrPro                              420425430                                                                     CCAGCACCTAAAGAAGATGATCCCCTTAAAAAATACACTTTTTGGGAA1344                          ProAlaProLysGluAspAspProLeuLysLysTyrThrPheTrpGlu                              435440445                                                                     GTAAATTTAAAGGAAAAGTTTTCTGCAGACCTAGATCAGTTTCCTTTA1392                          ValAsnLeuLysGluLysPheSerAlaAspLeuAspGlnPheProLeu                              450455460                                                                     GGACGCAAATTTTTACTACAAGCAGGATTGAAGGCCAAACCAAAATTT1440                          GlyArgLysPheLeuLeuGlnAlaGlyLeuLysAlaLysProLysPhe                              465470475480                                                                  ACATTAGGAAAACGAAAAGCTACACCCACCACCTCATCTACCTCTACA1488                          ThrLeuGlyLysArgLysAlaThrProThrThrSerSerThrSerThr                              485490495                                                                     ACTGCTAAACGCAAAAAACGTAAGCTGTA1517                                             ThrAlaLysArgLysLysArgLysLeu                                                   500505                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bovine papillomavirus                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: BPV1 N                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCGCTGAATTCAATATGGCGTTGTGGCAACAAGGCCAGAAGCTGTAT47                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: BPV1 Y                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCGGTGGTACCGTGCAGTTGACTTACCTTCTGTTTTACATTTACAGA47                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: HPV16 N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCGCTAGATCTAATATGTCTCTTTGGCTGCCTAGTGAGGCC41                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: HPV16 Y                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCGGTAGATCTACACTAATTCAACATACATACAATACTTACAGC44                                __________________________________________________________________________

What is claimed is:
 1. A method of detecting antibodies topapillomavirus in a specimen from a mammal comprising the steps of:(1)providing a self-assembled papillomavirus-like particle comprising a L1polypeptide having the same sequence as the amino acid sequence encodedby SEQ ID NO:2, wherein the self-assembled papillomavirus-like particlecomprises at least one papillomavirus L1 conformational epitope, andsaid self-assembled papillomavirus-like particle is produced by a methodcomprising the step of permitting a genetic construct, comprising apapillomavirus L1 gene, to direct recombinant expression in atransformed eukaryotic host cell; (2) contacting said specimen with saidself-assembled papillomavirus-like particle under conditions appropriatefor binding; and (3) detecting binding of said self-assembledpapillomavirus-like particle, where binding indicates the presence ofantibodies to papillomavirus in the specimen.
 2. The method of claim 1,wherein the self-assembled papillomavirus-like particle consists of theL1 polypeptide.
 3. The method of claim 1, wherein the self-assembledpapillomavirus-like particle further comprises a L2 polypeptide.
 4. Themethod of claim 1, wherein the self-assembled papillomavirus-likeparticle is made by expression of the L1 gene in an insect cell.
 5. Themethod of claim 1, wherein the self-assembled papillomavirus-likeparticle is made by expression of the L1 gene in a mammalian cell. 6.The method of claim 1, wherein the self-assembled papillomavirus-likeparticle is made by expression of the L1 gene in a yeast cell.
 7. Amethod of detecting antibodies to papillomavirus in a specimen from amammal comprising the steps of:(1) providing a self-assembledpapillomavirus-like particle ("VLP) comprising a L1 polypeptide, withthe proviso that said VLP does not contain a L2 polypeptide, said L1polypeptide having the same sequence as the amino acid sequence encodedby SEQ ID NO:2, wherein the self-assembled papillomavirus-like particlecomprises at least one papillomavirus L1 conformational epitope, andsaid self-assembled papillomavirus-like particle is produced by a methodcomprising the step of permitting a genetic construct, comprising apapillomavirus L1 gene, to direct recombinant expression in atransformed eukaryotic host cell; (2) contacting said specimen with saidself-assembled papillomavirus-like particle under conditions appropriatefor binding; and (3) detecting binding of said self-assembledpapillomavirus-like particle, where binding indicates the presence ofantibodies to papillomavirus in the specimen.
 8. A diagnostic kit fordetecting antibodies to papillomavirus in a specimen from a mammalcomprising:(1) a self-assembled papillomavirus-like particle comprisinga L1 polypeptide having the same sequence as the amino acid sequenceencoded by SEQ ID NO:2, wherein the self-assembled papillomavirus-likeparticle comprises at least one papillomavirus L1 conformationalepitope, and said self-assembled papillomavirus-like particle isproduced by a method comprising the step of permitting a geneticconstruct, comprising a papillomavirus L1 gene, to direct recombinantexpression in a transformed eukaryotic host cell; and (2) materials fordetecting binding between said self-assembled papillomavirus-likeparticle and antibodies to papillomavirus in said specimen, effective todetermine the presence or amount of said antibodies to papillomavirus,in a unit package container.
 9. A diagnostic kit for detectingpapillomavirus in a specimen from a mammal comprising:(1) aself-assembled papillomavirus-like particle ("VLP") comprising a L1polypeptide, with the proviso that said VLP does not contain a L2polypeptide, said L1 polypeptide having the same sequence as the aminoacid sequence encoded by SEQ ID NO:2, wherein the self-assembledpapillomavirus-like particle comprises at least one papillomavirus L1conformational epitope, and said self-assembled papillomavirus-likeparticle is produced by a method comprising the step of permitting agenetic construct, comprising a papillomavirus L1 genes to directrecombinant expression in a transformed eukaryotic host cell; and (2)materials for detecting binding between said self-assembledpapillomavirus-like particle and antibodies to papillomavirus in saidspecimen, effective to determine the presence or amount of saidantibodies to papillomavirus, in a unit package container.